PROJECT SUMMARY In the U.S. ~5 million individuals receive blood transfusions annually. Increasingly, these recipients are being put at risk of infection with Babesia microti, a tick-transmitted blood-borne parasite that resides in red blood cells (RBCs) and causes the disease babesiosis. B. microti represents a significant threat to the US blood supply as it survives blood banking procedures and storage conditions, and can be transmitted via transfusion. Transfusion transmitted babesiosis (TTB) is responsible for the highest percentage of transfusion-related infectious fatalities reported to the FDA. There is a critical unmet need for a method that is sensitive and specific, cost-effective, and high-throughput to screen donated blood for this pathogen. We propose the development of an EIA-based antigen-capture assay capable of detecting low levels of parasites in blood donors who are likely unaware that they are infected (e.g., asymptomatic, chronic carriers). We generated a library of antibodies using B. microti infected mice, and then screened this library to identify antibodies specific to parasite surface-expressed/secreted proteins. Using these methods, we have identified antibodies to seven B. microti proteins expressed during mammalian infection. In this study, we will generate an optimized antigen-capture assay using antibody pairs to one or more parasite secreted/surface target antigens that we have identified in preliminary studies. In Specific Aim 1, we will optimize capture and reporter antibody pairs for use in an antigen capture assay for the detection of B. microti, and confirm the specificity and sensitivity of these capture-reporter antibody pairs. In Specific Aim 2, we will performance our assay using blood obtained from patients with babesiosis, and identify a finalized antigen-capture assay to proceed to clinical assay screening with our commercialization partner in a phase II application.